Advancements in the understanding of gene expression and epidemiology combined with developments in technology have allowed for the correlation of genetic expression with, for example, disease states. An accurate correlation may enable risk assessment for an individual based on the expression profile of his/her individual cells or tissue. Further, drug screening and other research based protocols may quickly generate data in tissue samples that can be extended to develop treatments for human disease.
There is an abundance of specimens resulting from the pathological analysis of the disease; however, there are drawbacks to the typical methodologies available for evaluating them. For example, it is not always possible in the clinical setting to work on cell lines or tissue as soon as they are available and, consequently, they are often preserved in a fixative that permits the retention of cellular morphology and cellular constituents. The most common type of fixative is a cross-linking fixative that chemically couples proteins, RNAs, DNAs and small molecules in an insoluble matrix. Efforts have been made to isolate RNA from such fixed tissue in order to, for instance, analyze RNA populations. Some commercial products, for instance, Paradise™ Reagent System (Arcturus, Mountain View, Calif.), are available. These efforts, however, have universally found the RNA to be quite short. It is believed the short RNAs likely represent the regions of RNA between the chemical cross-linked residues. As a result of their short length, these short sequences are difficult to isolate and to analyze. Furthermore, amplification of these RNAs is often needed, and the amplification process is also impeded by the short length of the isolated RNAs.
Another drawback is that methods that require disaggregation of the sample, such as Southern, Northern, or Western blot analysis, are rendered less accurate by dilution of the malignant cells by the normal or otherwise non-malignant cells that are present in the same sample. Furthermore, the resulting loss of tissue architecture precludes the ability, for example, to correlate the presence of genetic abnormalities with malignant cells in a context that allows morphological specificity. This issue is particularly problematic in tissue types known to be heterogeneous, such as in human breast carcinoma, where a significant percentage of the cells present in any given area may be non-malignant.
In situ hybridization (ISH) is a powerful and versatile tool for the detection and localization of nucleic acids (DNA and RNA) within cell or tissue preparations. See, for instance, U.S. Pat. Nos. 5,750,340 and 6,165,723. In ISH, labeled nucleic acids (DNA or RNA) are hybridized to chromosomes, DNA or mRNAs in cells which are immobilized on microscope glass slides (In Situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In Situ Hybridization: In Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In Situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England (1992)). Numerous non-isotopic systems have been developed to visualize labeled DNA probes including, for example, a) fluorescence-based direct detection methods, b) digoxigenin- and biotin-labeled DNA probes coupled with fluorescence detection methods, and c) digoxigenin- and biotin-labeled DNA probes coupled with antibody-enzyme detection methods. When fluorescence-labeled nucleic acid (DNA or RNA) probes are hybridized to cellular DNA or RNA targets, the hybridized probes can be viewed directly using a fluorescence microscope. By using multiple nucleic acid probes with different fluorescence colors, simultaneous multicolored analysis (i.e., for multiple genes or RNAs) can be performed in a single step on a single target cell (Levsky et al., 2002, Science 297:836-840). Fluorochrome-directly labeled nucleic acid probes eliminate the need for multi-layer detection procedures (e.g., antibody-based system), which allows for faster processing and also reduces non-specific background signals. Therefore, fluorescence in situ hybridization (FISH) has become an increasingly popular and valuable tool in both basic and clinical sciences. ISH also been combined with polymerase chain reaction (PCR) to amplify, for instance, low abundance nucleic acid (see, e.g., Long, 1998, Eur. J. Histochem. 42:101-109). In situ PCR involves first amplifying, in situ, specific gene sequences, followed by in situ hybridization to detect the amplified sequences.
Through the use of labeled DNA or RNA probes, the ISH technique provides a high degree of spatial information in locating specific DNA or RNA target within individual cells or chromosomes. ISH is widely used for research and potentially for diagnosis in the areas of prenatal genetic disorders, and molecular cytogenetics. In the general area of molecular biology, ISH is used to detect gene expression, to map genes, to identify sites of gene expression, to localize target genes, and to identify and localize various viral and microbial infections. Currently, the application of the ISH technology research is being expanded into tumor diagnosis, preimplantation genetic diagnosis for in vitro fertilization, evaluation of bone marrow transplantation, and analysis of chromosome aneuploidy in interphase and metaphase nuclei.
U.S. Pat. No. 5,856,089 describes in situ hybridization methods using nucleic acid probes for single copy nucleic acid sequences to detect chromosomal structural abnormalities in fixed tissue obtained from a patient suspected of having a chromosomal structural abnormality. The methods include the use of bisulfite ion on the fixed cells.
U.S. Pat. No. 5,672,696 describes preparation of a sample for a gene analysis or high-purity nucleic acid suitable for gene amplification from a fixed, paraffin-embedded tissue sample comprising heating an aqueous suspension containing a surfactant having a protein-denaturation action and a deparaffinized tissue sample obtained from a paraffin-embedded tissue sample at 60° C. or higher.
U.S. Pat. No. 6,534,266 describes an in situ hybridization method for detecting and specifically identifying transcription of a multiplicity of different target sequences in a cell. The method includes assigning a different bar code to at least five target sequences, with each target sequence containing at least one, predetermined subsequence. Each bar code contains at least one fluorochrome, and at least one bar code comprises at least two different, spectrally-distinguishable fluorochromes. A probe set specific for each target sequence is provided in the method. Each probe set contains a hybridization probe complementary to each subsequence in the target sequence. Each probe is labeled with a fluorochrome, and the fluorochromes in each probe set collectively correspond to the bar code for the target sequence of that probe set.
U.S. Patent Application Publication No. 2005/0142589 discloses a method of detecting the relative amounts of multiple target messenger RNA's in a microscopy sample using in situ hybridization and sets of designed oligonucleotides probes. Each set comprises two or more oligonucleotides that will hybridize to a particular nucleic acid sequence of interest. The method permits the hybridization signal to be increased, for instance, for targets known or suspected to be in low abundance, by increasing the number of probes in a set that will hybridize to the target.
Spotted chip expression microarrays have been used extensively to detect the presence or absence of multiple specific mRNAs simultaneously in tissue. However, to date, the effective application of this technique has been limited to fresh frozen tissue. An easy application utilizing paraffin-embedded or other fixed-treated tissue spotted chip expression microarrays has not been described to date. See, for example, U.S. Patent Publication Nos. 20030040035 and 20020192702. Because many of the cell lines and tissue available for scientific or medical study have been fixed, the ability to effectively use spotted chip arrays on fixed-treated cell lines and tissue would be of great potential value in (1) the discovery of the molecular mechanisms of the cell and its surrounding tissue in health and disease, (2) the creation of tests diagnostic of disease, (3) the creation of treatments therapeutic for disease, and (4) the identification of agents that are toxic to cells.
There exists, therefore, a need in the art for a method utilizing fixed pathological specimens as a resource for studying disease. This need arises, in part, from the notion that an understanding of the gene expression patterns of certain diseases, such as cancer, may be central to successful management and treatment of diseases. The present invention fulfills this need by providing a procedure for the in situ cloning of RNAs from tissue specimens that have been fixed in any manner that retains RNA in a specimen.